Fluorescence Properties of Recombinant Tropomyosin Containing Tryptophan, 5-Hydroxytryptophan and 7-Azatryptophan
Kaustuv Das
Department of Chemistry, Iowa State University, Ames, IA, USA
Search for more papers by this authorKyle D. Ashby
Department of Chemistry, Iowa State University, Ames, IA, USA
Search for more papers by this authorAlex V. Smirnov
Department of Chemistry, Iowa State University, Ames, IA, USA
Search for more papers by this authorFernando C. Reinach
Departamento de Bioqufmica, Institute de Qufmica
Howard Hughes Medical Institute, Universidade de Sao Paulo, Sao Paulo, Brazil
Search for more papers by this authorJacob W. Petrich
Department of Chemistry, Iowa State University, Ames, IA, USA
*To whom correspondence should be addressed at: J.W.P.: Department of Chemistry, Iowa State University, Ames, IA 50011-3111 USA. Fax: 515-294-0105; e-mail:[email protected].C.S.F.: De-partamento de Bioqufmica, Instituto de Qufmica, Universidade de Sao Paulo, Sao Paulo, S.P., CEP 05508-900, Brazil. Fax: (+55-11)-815–5579; e-mail:[email protected]
Search for more papers by this authorChuck S. Farah
Departamento de Bioqufmica, Institute de Qufmica
*To whom correspondence should be addressed at: J.W.P.: Department of Chemistry, Iowa State University, Ames, IA 50011-3111 USA. Fax: 515-294-0105; e-mail:[email protected].C.S.F.: De-partamento de Bioqufmica, Instituto de Qufmica, Universidade de Sao Paulo, Sao Paulo, S.P., CEP 05508-900, Brazil. Fax: (+55-11)-815–5579; e-mail:[email protected]
Search for more papers by this authorKaustuv Das
Department of Chemistry, Iowa State University, Ames, IA, USA
Search for more papers by this authorKyle D. Ashby
Department of Chemistry, Iowa State University, Ames, IA, USA
Search for more papers by this authorAlex V. Smirnov
Department of Chemistry, Iowa State University, Ames, IA, USA
Search for more papers by this authorFernando C. Reinach
Departamento de Bioqufmica, Institute de Qufmica
Howard Hughes Medical Institute, Universidade de Sao Paulo, Sao Paulo, Brazil
Search for more papers by this authorJacob W. Petrich
Department of Chemistry, Iowa State University, Ames, IA, USA
*To whom correspondence should be addressed at: J.W.P.: Department of Chemistry, Iowa State University, Ames, IA 50011-3111 USA. Fax: 515-294-0105; e-mail:[email protected].C.S.F.: De-partamento de Bioqufmica, Instituto de Qufmica, Universidade de Sao Paulo, Sao Paulo, S.P., CEP 05508-900, Brazil. Fax: (+55-11)-815–5579; e-mail:[email protected]
Search for more papers by this authorChuck S. Farah
Departamento de Bioqufmica, Institute de Qufmica
*To whom correspondence should be addressed at: J.W.P.: Department of Chemistry, Iowa State University, Ames, IA 50011-3111 USA. Fax: 515-294-0105; e-mail:[email protected].C.S.F.: De-partamento de Bioqufmica, Instituto de Qufmica, Universidade de Sao Paulo, Sao Paulo, S.P., CEP 05508-900, Brazil. Fax: (+55-11)-815–5579; e-mail:[email protected]
Search for more papers by this authorAbstract
Tropomyosin mutants containing either tryptophan (122W), 5-hydroxytryptophan (50H122W) or 7-azatryp-tophan (7N122W) have been expressed in Escherichia coli and their fluorescence properties studied. The fluorescent amino acids were located at position 122 of the tropomyosin primary sequence, corresponding to a solvent-exposed position c of the coiled-coil heptapeptide repeat. The emission spectrum of the probe in each mutant is blue-shifted slightly with respect to that of the probe in water. The fluorescence anisotropy decays are single exponential, with a time constant of 2–3 ns while the fluorescence lifetimes of the probes incorporated into the proteins, in water, are nonexponential. Because tryptophan in water has an intrinsic nonexponential fluorescence decay, it is not surprising that the fluorescence decay of 122W is well described by a triple exponential. The fluorescence decays in water of the nonnatural amino acids 5-hydroxytryptophan and 7-azatryptophan (when emission is collected from the entire band) are single exponential. Incorporation into tropomyosin induces triple-exponential fluorescence decay in 5-hydroxytryptophan and double-exponential fluorescence decay in 7-azatryptophan. The range of lifetimes observed for 5-hy-droxyindole and 5-hydroxytryptophan at high pH and in the nonaqueous solvents were used as a base with which to interpret the lifetimes observed for the 50H122W and indicate that the chromophore exists in several solvent environments in both its protonated and unprotonated forms in 50H122W.
References
- 1 Petrich, J. W., M. C. Chang, D. B. McDonald and G. R. Fleming (1983) On the origin of nonexponential fluorescence decay in tryptophan and its derivatives. J. Am. Chem. Soc. 105, 3824–3832.
- 2 Chang, M. C., J. W. Petrich, D. B. McDonald and G. R. Fleming (1983) Nonexponential fluorescence decay of tryptophan, tryp-tophylglycine, and glycyltryptophan. J. Am. Chem. Soc. 105, 3819–3824.
- 3 Chen, L. X.-Q., J. W. Petrich, G. R. Fleming and A. Perico (1987) Picosecond fluorescence studies of polypeptide dynamics: fluorescence anisotropics and lifetimes. Chem. Phys. Lett. 139, 55–61.
- 4 Hogue, C. W. V., I. Rasquinha, A. G. Szabo and J. P. Mac-Manus (1992) A new intrinsic fluorescent probe for proteins: biosynthetic incorporation of 5-hydroxytrytophan into oncom-odulin. FEBS Lett. 310, 269–272. [Abstract].
- 5 Szabo, A. G. and D. M. Rayner (1980) Fluorescence decay of tryptophan conformers in aqueous solution. J. Am. Chem. Soc. 102, 554–563.
- 6 Szabo, A. G. and D. M. Rayner (1980) The time-resolved emission spectra of peptide conformers measured by pulsed laser excitation. Biochem. Biophys. Res. Commun. 94, 909–915.
- 7 Cockle, S. A. and A. G. Szabo (1981) Time-resolved fluorescence spectra of tryptophan in monomeric glucagon. Photo-chem. Photobiol. 34, 23–27.
- 8 Werner, T. C. and L. S. Forster (1979) The fluorescence of tryptophyl peptides. Photochem. Photobiol. 29, 905–914.
- 9 Ross, J. B., K. W. Rousslang and L. Brand (1981) Time-resolved fluorescence and anisotropy decay of tryptophan in ad-renocorticotropin-(l-24). Biochemistry 20, 4361–4369.
- 10 Creed, D. (1984) The photophysics and photochemistry of the near-UV absorbing amino acids–I. Tryptophan and its simple derivatives. Photochem. Photobiol. 39, 537–562.
- 11 Beechem, J. M. and L. Brand (1985) Time-resolved fluorescence of proteins. Annu. Rev. Biochem. 54, 43–71.
- 12 Chen, Y., F. Gai and J. W. Petrich (1994) Single-exponential fluorescence decay of the nonnatural amino acid 7-azatrypto-phan and the nonexponential fluorescence decay of tryptophan in water. J. Phys. Chem. 98, 2203–2209.
- 13 Negrerie, M., S. M. Bellefeuille, S. Whitham, J. W. Petrich and R. W. Thornburg (1990) Novel noninvasive in situ probe of protein structure and dynamics. J. Am. Chem. Soc. 112, 7419–7421. [ABSTRACT].
- 14 Chen, Y., F. Gai and J. W. Petrich (1993) Solvation of 7-azain-dole in alcohols and water: evidence for concerted, excited-state, double-proton transfer in alcohols. J. Am. Chem. Soc. 115, 10158–10166.
- 15 Rich, R. L., Y. Chen, D. Neven, M. Ndgrerie and J. W. Petrich (1993) Steady-state and time-resolved fluorescence anisotropy of 7-azaindole and its derivatives. J. Phys. Chem. 97, 1781–1788.
- 16 Chen, Y., R. L. Rich, F. Gai and J. W. Petrich (1993) Fluorescent species of 7-azaindole and 7-azatryptophan in water. J. Phys. Chem. 97, 1770–1780.
- 17 Smirnov, A. V., R. L. Rich, and J. W. Petrich (1994) Synthesis and spectral characterization of 5′-phosphopyridoxyl-D, L-7-aza-tryptophan, a photophysical probe of protein structure and dynamics. Biochem. Biophys. Res. Commun. 198, 1007–1011.
- 18 Gai, F., Y. Chen and J. W. Petrich (1992) Nonradiative pathways of 7-azaindole in water. J. Am. Chem. Soc. 114, 8343–8345.
- 19 Rich, R. L., A. V. Smirnov, A. W. Schwabacher and J. W. Petrich (1995) Synthesis and photophysics of the optical probe N, -methyl-7-azatryptophan. J. Am. Chem. Soc. 117, 11850–11853.
- 20 Rich, R. L., M. Negrerie, J. Li, S. Elliot, R. W. Thornburg and J. W. Petrich (1993) The photophysical probe, 7-azatryptophan, in synthetic peptides. Photochem. Photobiol. 58, 28–30.
- 21 Rich, R. L., F. Gai, J. W. Lane, J. W. Petrich and A. W. Schwabacher (1995) Using 7-azatryptophan to probe small molecule-protein interactions on the picosecond time scale: the complex of avidin and biotinylated 7-azatryptophan. J. Am. Chem. Soc. 117, 733–739.
- 22 English, D. S., R. L. Rich and J. W. Petrich (1998) Nonexponential fluorescence decay of 7-azatrytophan induced in a peptide environment. Photochem. Photobiol. 67, 76–83.
- 23 Smirnov, A. V., D. S. English, R. L. Rich, J. Lane, L. Teyton, A. W. Schwabacher, S. Luo, R. W. Thornburg and J. W. Petrich (1997) Photophysics and biological applications of 7-azaindole and its analogs. J. Phys. Chem. B 101, 2758–2769.
- 24 Smillie, L. B. (1979) Structure and functions of tropomyosins from muscle and non-muscle sources. Trends Biochem. Sci. 4, 151–155.
- 25 Lupas, A. (1996) Coiled coils: new structures and new functions. Trends Biochem. Sci. 21, 375–382.
- 26 Zot, A. S. and J. D. Potter (1987) Structural aspects of troponin-tropomyosin regulation of skeletal muscle contraction. Annu. Rev. Biophys. Chem. 16, 535–559.
- 27 Chalovich, J. M. (1992) Actin-mediated regulation of muscle contraction. Pharmacol. Ther. 55, 95–148.
- 28 Farah, C. S. and F. C. Reinach (1995) The troponin complex and regulation of muscle contraction. FASEB J. 9, 755–767.
- 29 Tobacman, L. S. (1996) Thin filament-mediated regulation of cardiac contraction. Annu. Rev. Physiol. 58, 447–481.
- 30 Huxley, H. E. (1972) Structural changes in the actin- and myosin-containing filaments during contraction. Cold Spring Harbor Symp. Quant. Biol. 37, 361–376.
- 31 Lehman, W., R. Craig and P. Vibert (1994) Ca2+-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature 368, 65–67.
- 32 Holmes, K. C. (1995) The actomyosin interaction and its control by tropomyosin. Biophys. J. 68, 2S–7S.
- 33 Ross, J. B. A., A. G. Szabo and C. W. V. Hogue (1997) Enhancement of protein spectra with tryptophan analogs: fluorescence spectroscopy of protein-protein and protein-nucleic acid interactions. Methods Enzymol. 278, 151–190.
- 34 Wong, C.-Y. and M. R. Eftink (1997) Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability. Protein Sci. 6, 689–697.
- 35 Hogue, C. W. V. and A. G. Szabo (1993) Characterization of aminoacyl-adenylates in B. subtilis tryptophanyl-tRNA synthetase, by the fluorescence of tryptophan analogs 5-hydroxytryptophan and 7-azatryptophan. Biophys. Chem. 48, 159–169 (Abstract).
- 36 Monteiro, P. B., R. C. Lataro, J. A. Ferro and F. De Castro Reinach (1994) Functional a-tropomyosin produced in Escherichia coli: a dipeptide extension can substitute the amino-ter-minal acetyl group. J. Biol. Chem. 269, 10461–10466.
- 37 Bevington, P. R. and D. K. Robinson (1992) Data Reduction and Error Analysis for the Physical Sciences, 2nd ed. pp. 65–72. McGraw-Hill, New York .
- 38 Chang, Y.-C. and R. D. Ludescher (1994) Tryptophan photophysics in rabbit skeletal myosin rod. Biophys. Chem. 49, 113–126.
- 39 Ruggiero, A. J., D. C. Todd and G. R. Fleming (1990) Subpi-cosecond fluorescence anisotropy studies of tryptophan in water. J. Am. Chem. Soc. 112, 1003–1014.
- 40 Chen, L. X.-Q., R. A. Engh and G. R. Fleming (1988) Reorientation of tryptophan and simple peptides: onset of internal flexibility and comparison with molecular dynamics simulation. J. Phys. Chem. 92, 4811–4816.
- 41 Eftink, M. R., L. A. Selvidge, P. R. Callis and A. A. Rehms (1990) Photophysics of indole derivatives: experimental resolution of La and Lb transitions and comparison with theory. J. Phys. Chem. 94, 3469–3479.
- 42 Callis, P. R. (1997) La and Lb transitions of tryptophan: applications of theory and experimental observations to fluorescence of proteins. Methods Enzymol. 278, 113–150.
- 43 Cross, A. J., D. H. Waldeck and G. R. Fleming (1983) Time resolved polarization spectroscopy: level kinetics and rotational diffusion. J. Chem. Phys. 78, 6455–6465.
- 44 Valeur, B. and G. Weber (1977) Resolution of the fluorescence excitation spectrum of indole into the La and Lb excitation bands. Photochem. Photobiol. 25, 441–444.
- 45 Wahl, P., K. Tawada and J.-C. Auchet (1978) Study of tropomyosin labeled with a fluorescent probe by pulse fluorimetry in polarized light. Eur. J. Biochem. 88, 421–424.
- 46 Holtzer, M. E., D. L. Crimmins and A. Holtzer (1995) Structural stability of short sequences of the tropomyosin chain. Biopoly-mers 35, 125–136.
- 47 Privalov, P. L. (1982) Stability of proteins: proteins which do not present a single cooperative system. Adv. Protein Chem. 35, 1–104.
- 48 Meech, S. R., D. Phillips and A. G. Lee (1983) On the nature of the fluorescent state of methylated indole derivatives. Chem. Phys. 80, 317–328.
- 49 Udenfriend, S., H. Weissbach and B. B. Brodie (1958) Assay of serotonin and related metabolites, enzymes, and drugs. Methods Biochem. Anal. 6, 95–130.
- 50 Whitby, F. G., H. Kent, F. Stewart, M. Stewart, X. Xie, V. Hatch, C. Cohen and G. N. Phillips, Jr. (1992) Structure of tropomyosin at 9 Angstroms resolution. J. Mol. Biol. 227, 441–452.
- 51 Phillips, G. N., Jr. (1986) Appendix: construction of an atomic model for tropomyosin and implications for interactions with actin. J. Mol. Biol. 192, 128–131.
- 52 Gooding, C., F. C. Reinach and A. R. MacLeod (1987) Complete nucleotide sequence of the fast-twitch isoform of chicken skeletal muscle α-tropomyosin. Nucleic Acids Res. 15, 8105.
- 53 Maliwal, B. P., A. D. Cardin, R. L. Jackson and J. R. Lakowicz (1985) Nanosecond motions of the single tryptophan residues in apolipoproteins C-I and C-II: a study by oxygen quenching and fluorescence depolarization. Arch. Biochem. Biophys. 236, 370–378.
- 54 Tao, T. and J. Cho (1979) Fluorescence lifetime quenching studies on the accessibilities of actin sulfhydryl sites. Biochemistry 18, 2759–2765.
- 55 Ricchelli, F., M. Beltramini, L. Flamigni and B. Salvato (1987) Emission quenching mechanisms in Octopus vulgaris hemocy-anin: steady-state and time-resolved fluorescence studies. Biochemistry 26, 6933–6939.
- 56 Junius, F. K., S. I. O'Donoghue, M. Nilges, A. S. Weiss and G. F. King (1996) High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer. J. Biol. Chem. 271, 13663–13667.
- 57 Kishi, T., M. Tanaka and J. Tanaka (1977) Electronic absorption and fluorescence spectra of 5-hydroxytryptamine (serotonin). Protonation in the excited state. Bull. Chem. Soc. Jpn. 50, 1267–1271.
- 58 Forster, T. (1965) Delocalized excitation and excitation transfer. In Modern Quantum Chemistry. Part III: Action of Light and Organic Crystals (Edited by O. Sinanoglu), pp. 93–137. Academic Press, New York .
- 59
Lakowicz, J. R. (1983) Energy transfer. In
Principles of Fluorescence Spectroscopy, pp.
303–339. Plenum Press,
New York
.
10.1007/978-1-4615-7658-7_10 Google Scholar
